THYROTROPIN-RELEASING HORMONE RECEPTOR MOLECULAR BIOLOGY

该研究计划的广泛、长期目标是定义 促甲状腺素释放激素（TRH）的作用机制，以便更好地 了解所有细胞外信号调节细胞的机制 分子，例如激素、神经递质和生长因子 一些扰乱细胞调节的病理过程。 具体来说， 垂体前叶细胞 TRH 受体的分子生物学将 研究过。 TRH 受体的互补 DNA (cDNA) 几乎已 被克隆了。 克隆策略涉及功能性 TRH 的表达 来自小鼠垂体促甲状腺激素生成 (TtT) 的受体 非洲爪蟾卵母细胞中的肿瘤。 我们首先建议对 cDNA 进行测序并 然后使用它和推导的受体蛋白的氨基酸序列 研究确定 TRH 受体的组织分布 TRH 受体的结构与功能关系，特别是 TRH 受体关系的分子细节，特别是 它与 TRH 相互作用及其与鸟嘌呤偶联的分子细节 核苷酸结合调节（G）蛋白及其调节机制 TRH 受体数量，这是经过验证的 TRH 调节机制 行动。 将使用 GH3 大鼠垂体和大鼠进行研究 组织。 使用双脱氧核苷酸方法的测序策略已被 设计的。 将使用放射性标记的 cDNA 进行原位杂交 探针。 将产生 cDNA 的选择性突变，然后用于 转化 GH-Y 细胞，GH3 的亚克隆，无 TRH 受体或可检测 受体 mRNA 活性，定义 TRH 的功能域 受体。 并且，TRH受体数量的调节机制将是 通过测量其水平以及合成和降解率来研究 受体蛋白和 mRNA，以及 mRNA 活性的互补测量 在非洲爪蟾卵母细胞中制备以确定是否有变化 翻译效率。 TRH 受体的磷酸化 作为其调节的可能机制进行研究。

The broad, long term objective of this research program is to define the mechanism of action of thyrotropin-releasing hormone (TRH) so as to better understand the mechanism of cell regulation by all extracellular signal molecules, such as hormones, neurotransmitters and growth factors and of some pathologic processes that disorder cell regulation. Specifically, the molecular biology of the TRH receptor from anterior pituitary cells will be studied. A complementary DNA (cDNA) for the TRH receptor has very nearly been cloned. The cloning strategy involved expression of a functional TRH receptor from a mouse pituitary thyroid-stimulating hormone-producing (TtT) tumor in Xenopus laevis oocytes. We first propose to sequence the cDNA and then use it and the deduced amino acid sequence of the receptor protein in studies to define the tissue distribution of the TRH receptor, the structure-function relations of the TRH receptor, in particular the molecular details of its relations of the TRH receptor, in particular the molecular details of its interaction with TRH and its coupling to a guanine nucleotide binding regulatory (G) protein, and the mechanism of modulation of TRH receptor number, which is a proven mechanism for regulation of TRH action. Studies will be performed using GH3 rat pituitary glands and rat tissues. A sequencing stategy using the dideoxynucleotide method has been devised. In situ hybridization will be performed with radiolabeled cDNA probes. Selective mutations of the cDNA will be made and then used to transform GH-Y cells, a subclone of GH3 without TRH receptors or detectable receptor mRNA activity, to define the functional domains of the TRH receptor. And, the mechanism of regulation of TRH receptor number will be studied by measuring the levels and synthesis and degradation rates of the receptor protein and mRNA, with complementary measurements of mRNA activity made in Xenopus oocytes to determine whether there are changes in translational efficiency. Phosphorylation of the TRH receptor will be studied as a possible mechanism of its regulation.